CN111979357A - Detection method of bovine viral diarrhea virus based on CRISPR-Cas13a - Google Patents

Abstract

The invention discloses a primer for detecting bovine viral diarrhea virus, a kit for detecting BVDV prepared by using the primer, and a method for detecting the bovine viral diarrhea virus based on CRISPR-Cas13a, wherein the primer comprises the following components: 5'-GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCCAUCCAACGAACUCACCACUGUUGCU-3', respectively; the kit at least comprises the primer, and a sample to be detected is added into the kit for detection. According to the invention, the CRISPR-Cas13a system is successfully used for the nucleic acid detection of the BVDV virus for the first time, and whether the BVDV virus is contained can be identified only by adding a micro-scale sample. Has good specificity and sensitivity. The method provides a wide prospect for early diagnosis of BVDV.

Description

Detection method of bovine viral diarrhea virus based on CRISPR-Cas13a

Technical Field

The invention relates to the technical field of biological detection, in particular to a method for detecting bovine viral diarrhea virus based on CRISPR-Cas13a.

Background

Bovine Viral Diarrhea (mucosal disease) is a contact infectious disease caused by Bovine Viral Diarrhea Virus (BVDV), mainly infects cattle, and also infects other organisms such as pigs, sheep, camels, etc., and the host range is wide. BVDV is a positive sense ribonucleic acid (RNA) virus belonging to the flaviviridae and pestivirus genera. Disorders that can lead to a variety of clinical manifestations after infection, including respiratory, digestive and reproductive (in various forms) (ii)

B,Roch F F,Richter V,et al.A meta-analysis of bovine viral diarrhoea virus(BVDV)prevalences in the global cattle population[J]Scientific reports,2018,8(1): 1-15.). The traditional Chinese medicine composition is mainly used for treating acute or chronic mucosal diseases, diarrhea, growth and reproduction disorder, immunosuppression, persistent infection and other symptoms of cattle and sheep, once infection occurs, the growth, development and reproduction of the cattle are directly affected, and the immunity is partially reduced, so that infection of other pathogens can be induced. The BVDV has extremely wide hosts, and most wild animals can be infected with the BVDV and can carry and expel toxin, so that the probability of BVDV infection of the free-range animals is increased. Therefore, BVDV seriously threatens the development of livestock husbandry in China and even in the world, and causes great economic loss in the world. Therefore, detection and prevention of BVDV has very important significance (Garousi M T, Mehrzad J, Nejati A. invasion of persistent infection of Bone Viral Diarrhea Virus (BVDV) in Holstein days [ J]A pharmaceutical animal health and production,2019,51(4): 853-. Currently, there are many methods for detecting BVDV virus, including traditional serum neutralization assays, immunohistochemistry, ELISA and more recently fluorescent PCR techniques. However, conventional BVDV methods almost require sophisticated instrumentation and are costly, difficult to handle on-site and large-scale detection, and have a high probability of false positives (Adams M J, Hendrickson R C, Dempsey D M, et al].Archives of virology,2015,160(5):1375-1383.). Therefore, a method for detecting BVDV quickly, accurately and efficiently is urgently needed to be established.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) developed in recent years is an excellent genome editing tool, and infinite scientific research progress and application updating thereof cause revolutionary storms to the biological world, which also brings huge application prospects to the aspect of virus detection. In 2015, the structure of C2C2(Cas13a) was found: comprising a recognition domain and two HEPN ribonuclease domains (Shmakov, S., Abudayyeh, O.O., Makarova, K.S., Wolf, Y.I., Gootenberg, J.S., Semenova, E., … Koonin, E.V. (2015) Discovery and Functional Characterization of variant Class 2 CRISPR-systems. molecular Cell,60(3), 385-). Subsequently, it was found that these Two domains have Two different RNase activities, one on the guide RNA (gRNA) and the other on the target RNA (East-Seletsky, Alexandra, et al. "Two distintint RNase activities of CRISPR-C2C2 enable guide-RNA processing and RNA detection." Nature 538.7624(2016): 270-273.).

Further exploration shows that the crRNA is complementary to the target RNA to form double strand and then can be combined with nucleic acid lobe (NUC), and the combination makes Cas13a undergo significant conceived change. The formation of the RNA duplex triggers the HEPN1 to move to the HEPN2 domain, activating the HEPN catalytic site of the Cas13a protein, causing it to cleave single-stranded targets and other RNAs in a non-specific manner (Goodenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, et al. nucleic acid detection with CRISPR-Cas13 a/C2. science.2017; 356: 438-42.). The non-target (side effect) cleavage activity after the specific recognition of nucleic acid shows that the CRISPR/Cas biology is expected to be a rapid, accurate and portable diagnostic tool. Gootenberg et al have reported that Leptotrichia wadei Cas13a (LwCas13a) activates RNase activity to detect Zika virus and dengue virus in a sample (Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. science.2018; 360: 439-44.). Yafei et al also specifically detect PRRS using CRISPR-Cas13a (Chang Y, Deng Y, Li T, et al. visual detection of a positive reproduction and reproduction synthesis using CRISPR-Cas13a. [ J ]. Transboundary and electronic Diseases,2020,67(2): 564-571.). With the continuous development of detection technology, the superiority of the detection method by using the CRISPR-Cas13a is gradually highlighted. The CRISPR-Cas13a detection platform was developed as a new strategy for the detection of RNA viral diseases.

Disclosure of Invention

The invention aims to provide a method for quickly detecting BVDV, which is based on good specificity and high sensitivity of CRISPR-Cas13a.

Aiming at the problems mentioned in the background technology, the invention adopts the technical scheme that:

a method for detecting bovine viral diarrhea virus based on CRISPR-Cas13a comprises the steps of constructing a prokaryotic expression vector LwCas13a, carrying out inducible expression and purification on LwCas13a, designing specific crRNA, extracting BVDV virus RNA, establishing a CRISPR-Cas13a detection system and judging results, and comprises the following specific steps:

construction of a LwCas13a prokaryotic expression vector: the LwCas13a full-length gene is obtained by PCR and cloned to pET-28a by an enzyme digestion connection method⁺Construction of pET-28a on the vector⁺-LwC13a recombinant expression vector;

inducible expression purification of LwCas13 a: the prokaryotic expression vector is taken and transferred into Rosetta^TM(DE3) followed by expansion and expression induced by IPTG at low temperature (18 ℃ C.). Collecting cell precipitates, carrying out ultrasonic disruption after overnight lysis, taking the supernatant after ultrasonic disruption, obtaining LwCas13a protein by a nickel column affinity chromatography according to the instruction step GE, carrying out SDS-PAGE verification, and storing at-80 ℃ for later use after determining the protein concentration;

design of specific crRNA: and logging in NCBI, selecting a 5' UTR conserved region according to gene sequences (sequence numbers: KF501393.1, U63479.1 and AJ133738.1) in GenBank, screening, searching for a highly conserved site, and designing crRNA. The sequence is as follows:

5’-GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCCAUCCAACGAACUCACCACUGUUGCU-3’；

the quenching fluorescence reporter molecule RNA sequence is:

5'-GAAGAAGAGUUUAUUCAGAUAGAUUUGU-3', modifying the 5 'end into FAM and the 3' end into BHQ-1;

extraction of BVDV viral RNA: firstly culturing MDBK cells, infecting BVDV virus when the cell fusion degree reaches to be close to 70%, then continuing culturing for 72-96 h, and collecting BVDV virus liquid when MDBK cytopathy is close to 80%. Extracting BVDV virus RNA by using a QIAamp Viral RNA Mini Kit, detecting the extracted RNA by agarose gel electrophoresis and a trace nucleic acid protein detector (ND2000) to determine the purity of the RNA, and storing the RNA in an environment at-20 ℃ for later use;

establishment of CRISPR-Cas13a detection system: and uniformly mixing the 45nM purified LwCas13a protein, 22.5nM specific crRNA, 125nM quenched fluorescent reporter RNA, 2uLRNase inhibitor and detection buffer solution in a 96-well plate to obtain a mixture in a total volume of 40uL, and detecting on a Synergy HTX multifunctional microplate detector. Fluorescence was measured under the following conditions: FAM channels (. lamda.ex 485nm,. lamda.em 518nm), kinetic assay at 37 ℃ for 1h, measured every 5 min.

And (5) judging a result: when a sample to be detected contains BVDV virus RNA, a crRNA sequence can be combined with a designated target RNA, the target RNA is recognized by Cas13a, the conformation of the target RNA is changed, the RNase enzyme cleavage activity is activated, and meanwhile, the target RNA enters an enzymatic 'activated' state, and fluorescence is released by quenching fluorescence reporter RNA in a non-specific cleavage system. The method has the advantages of high specificity, good sensitivity and repeatability, simplicity, rapidness, high application value and popularization prospect.

The CRISPR-Cas13a detection system can obtain a result within 1 hour only by a micro-scale sample, so compared with other molecular biological gene diagnosis methods, the method has the characteristics of specificity, rapidness, accuracy and simplicity. The system utilizes the auxiliary activity of Cas13a, and quenched fluorescent reporter RNA is introduced into the system, which does not emit fluorescence generally, but if target RNA is contained in the system, crRNA binds to the target position, and Cas13a is guided to recognize and cleave the target RNA designated by the crRNA sequence. Simultaneously, the accessory activity of Cas13a, namely the exposed HEPN catalytic site can cut other non-target RNA in the system, and then the quenching fluorescent RNA reporter molecule in the system is cut to release fluorescence, so that the sample contains BVDV.

Compared with the prior art, the invention has the beneficial effects that:

1) the detection method has the advantages of good specificity, high sensitivity, good repeatability, high accuracy, capability of quickly detecting the BVDV, low detection cost, higher application value and popularization prospect;

2) the detection method of the invention designs BVDV specific crRNA, which can form complementary double-strand with BVDV conserved sequence specificity, and has no cross reaction with other viruses or cells, thus improving the BVDV detection specificity.

3) According to the detection method, quenching fluorescent reporter molecule RNA is introduced into the system, and detection signals in the system can be amplified by using the activity of the accessory RNA enzyme of Cas13a, so that the sensitivity in the detection system reaches 10³pM。

Drawings

FIG. 1 recombinant expression vector pET-28a⁺LwCas13a restriction electrophoretogram.

Figure 2 results of low temperature induction of LwCas13a protein expression.

Fig. 3LwCas13a protein purification.

FIG. 4 shows the detection of different viruses and cells by the method of the invention, i.e.the detection is specific to the method of the invention.

FIG. 5 is a test picture obtained by detecting BVDV RNA after gradient dilution by the method of the present invention, i.e., the detection of sensitivity by the method of the present invention, wherein the sensitivity of the method of the present invention is 10³pM。

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The inventor takes LwCas13a as a research center to express and purify LwCas13a protein, and designs a section of specific crRNA according to a reported conserved region of 5' UTR of a BVDV gene sequence. The BVDV-obtained RNA is used for verifying that Cas13a also has RNA enzyme digestion activity in vitro, and BVDV can be specifically and accurately detected, thereby completing the invention.

The first step is as follows: primer design of bovine viral diarrhea virus detection method based on CRISPR-Cas13a

LwCas13a gene primer design

The full length of the gene sequence of pC013-Twinstrep-SUMO-LwCas13a is obtained on addgene, and the full-length primer of LwCas13a protein is designed by using Premier 5 software. The primer sequence is as follows,

the forward primer F is 5'-CATGCCATGGGCAGCAGCATGAAAGTGACCAAGGTCGACG-3', and the reverse primer F is 5'-CATGCCATGGGCAGCAGCATGAAAGTGACCAAGGTCGACG-3',

the reverse primer R is 5'-GGCGAGCTCCCTTCCAGGGCCTTGTACTCGA-3', and the reverse primer R is 5'-GGCGAGCTCCCTTCCAGGGCCTTGTACTCGA-3',

the expected amplified fragment size was 3491 bp.

Design of crRNA

The NCBI is registered, and according to the gene sequences (sequence numbers: M31182, KF501393.1, U63479.1 and AJ133738.1) in GenBank, sequences of genes containing a highly conserved region of the 5' UTR region are selected and aligned, a highly conserved site is searched, and crRNA is designed. The sequence is as follows:

5’-GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCCAUCCAACGAACUCACCACUGUUGCU-3’。

BVDV primer design

Designing a detection primer according to a BVDV gene sequence, designing a pair of specific identification primers in a conserved region of the BVDV, wherein the primer sequence is,

a forward primer F: 5'-ATGCCCTTAGTAGGACTAGCA-3';

5'-TCAACTCCATGTGCCATGTAC-3', the expected amplified fragment size is 288 bp.

4. Quenching fluorescent reporter RNA sequence

Reference (Gootenberg, J.S., Abudayyeh, O.O., Lee, J.W., Essletzbichler, P., Dy, A.J., Joung, J.Zhang, F. (2017). Nucleic acid detection with CRISPR-Cas13 a/C2. science,356(6336), 438-.

The sequence is as follows: 5'-GAAGAAGAGUUUAUUCAGAUAGAUUUGU-3', the 5 'end is modified to FAM and the 3' end is modified to BHQ-1.

The second step is that: expression and purification of LwCas13a protein in prokaryotic system

Construction of LwCas13a prokaryotic expression vector

Designing and synthesizing a characteristic primer according to the LwCas13a full-length gene, obtaining the LwCas13a full-length gene sequence through PCR and mixing the sequence with a vector pET-28a⁺(ThermoFisher Scientific, Invitrogen) was subjected to sexual double digestion with NcoI (Takara, Dalian, China) and Xho I (Takara, Dalian, China), respectively, purified and recovered, and ligated at 16 ℃ using DNA ligase to transform DH 5. alpha. competence. After enzyme digestion identification, the obtained recombinant vector is named as pET-28a⁺-LwCas13a。

Expression of LwCas13a protein

The recombinant plasmid pET-28a obtained in the step 1 is⁺Transformation of LwCas13a into Rosetta^TM(DE3) pLysS competence. After screening and identification, the cells were expanded in LB liquid culture and then transferred to TB medium, and grown at 37 ℃ and 200rpm to OD600 of 0.5. At this time, expression was induced at 18 ℃ for 20h by supplementation with IPTG (500uM isopropyl-1-thio-. beta. -D-galactopyranoside) to a final concentration of 500 uM.

Purification of LwCas13a protein

Centrifuging the bacterial solution obtained in step 2 at 4 deg.C and 8000g for 15min to collect cell precipitate, and treating with lysis buffer (20mM NaH)₂PO₄300mM NaCL, 10mM imidazole pH 8.0) resuspend the cell pellet and lyse overnight. Repeatedly freezing and thawing the lysed cell sap in liquid nitrogen and a constant-temperature water bath at 37 ℃ for three times, and then carrying out ultrasonic treatment (ultrasonic conditions: 3s on and 2s off and 60% of power) by using an ultrasonic cell crusher (Ningbo Xinzhi Scienz-IID) until the cell sap is clear. Thereafter, the lysate was separated by centrifugation at 12000g for 40min at 4 ℃ to obtain a supernatant and a precipitate. The pellet was resuspended in 8M urea and protein expression was tested in the supernatant by SDS-PAGE and Coomassie blue staining.

The supernatant was filtered through a 0.22um filter, and then loaded onto 5mL of HisTratpTM FF crud (GE Healthcare Life Sciences) and subjected to the procedures described in the specification to obtain a protein elution buffer. The purification of the protein of interest in the fractions obtained was tested by SDS-PAGE and concentrated by ultrafiltration centrifuge tubes (Millipore) into protein storage buffer (600mM NaCl, 50mM Tris-HCl pH 7.5, 5% glycerol, 2mM DTT) and stored frozen at-80 ℃ after determination of the protein concentration using the BCA (bicinchoninic acid) protein quantification kit.

The third step: virus culture and RNA preparation

1. Culture of cells and viruses

Recovering MDBK cells and HEK293T cells in DMEM cell culture solution supplemented with 10% fetal bovine serum FBS, shaking to uniform at 37 deg.C and 5% CO₂The incubator is used for culture. After the cells were spread over the whole plate, the culture medium was discarded, washed 2 times with PBS, digested for 1min with pancreatin (containing 0.02% EDTA-2Na and 0.25% trypsin), and the cells were collected and stored at-80 ℃ for further use. And (2) additionally taking MDBK cells in logarithmic growth phase for passage, discarding culture solution when the cell fusion degree is about-70%, cleaning by PBS, adding new culture solution and BVDV virus suspension (NADL strain), adsorbing for 2h, adding 2% FBS DMEM culture solution, continuing conventional culture until more than 80% of cells are pathologically changed, and harvesting: freeze thawing the pathological cell culture plate for 3-5 times repeatedly, shaking to completely drop cells, collecting culture solution, centrifuging, collecting supernatant, and storing at-80 deg.C.

2. Extraction of cellular and viral RNA

MDBK cells and HEK293T cells were used to extract total RNA using an rnample total RNA extraction kit (QIAGEN, Duesseldorf, Germany) according to the instructions. BVDV virus fluid was prepared by extracting RNA using QIAamp Viral RNA Mini Kit (QIAGEN, Duesseldorf, Germany) according to the instructions and storing at-80 ℃ for use.

The fourth step: establishment of CRISPR-Cas13a detection system

CRISPR-Cas13a assay systems CRRNA (22.5nM), quenched fluorescent reporter RNA (125nM), LwCas13a protein (45nM), viral RNA (100ng), RNase inhibitor (New England Biolabs) (2uL), assay buffer (40mM Tris-HCl, 60mM NaCl, 6mM MgCl. sub.13) obtained in examples 1, 2, and 3₂pH 7.3). The above components (total volume 40 μ L) were added to a 96-well plate (corning 3915) and mixed uniformly, and then put in a Synergy HTX multifunctional microplate detector (BioTek) as followsConditions measurement fluorescence: excitation wavelength 485nm, emission wavelength 518nm, fluorescence kinetics were measured every 5 minutes for 1 hour at 37 ℃ and each treatment was repeated three times.

The fifth step: detection of specificity and sensitivity of the method of the invention

1. Detection of specificity of the methods of the invention

The RNA of the MDBK cell, HEK293T cell and BVDV virus solution obtained in example 3 were selected as the detection targets, and RNase-free H was added₂And (3) taking O as a negative control and taking BVDV RNA as a positive control to verify the specificity of the method. The fluorescence signal increased with time when BVDV RNA and BVDV virus fluid were added, the latter increased at a significantly higher rate than the former, while the negative control and others did not have significant difference. The detection method is used for identifying the crRNA of the BVDV virus RNA, has good atopy, and can distinguish the BVDV RNA or the BVDV virus liquid.

2. Detection of sensitivity of the method of the invention

BVDV virus solution obtained in example 3 was selected and extracted with QIAamp Viral RNA Mini Kit to obtain BVDV virus RNA as a sample. And (3) making a standard curve for the standard RNA of the BVDV by using reverse transcription fluorescence quantitative PCR, and detecting the RNA concentration of the extracted BVDV virus sample by comparing the standard curve. The test samples were diluted according to a 10-fold gradient dilution method, 8 sets of standards were set, and 3 duplicate wells were each set to evaluate the sensitivity of this experimental method. The BVDV RNA concentration which can be detected by the method is 10³PM。

The CRISPR-Cas13a detection method can obtain results within 1h, and has the characteristics of simplicity, convenience, rapidness, accuracy and specificity compared with other traditional biological diagnosis. The principle is as follows: the activity of the LwCas13a enzyme was exploited. Cas13a contains a REC domain, which is responsible for the processing and maturation of pre-crRNA, and a nuclease recognition domain, NUC, domain, which undergoes a conformational change upon binding to crRNA. When BVDV viral RNA is added to the system and the crRNA binds to the BVDV target site to form a double-stranded structure, Cas13a can recognize and cleave the target RNA designated by the crRNA sequence and enter an enzymatic "activated" state, where it can bind to and cleave other non-target RNAs in the system, which causes the quenched fluorescent RNA reporter present in the system to be cleaved and fluoresce.

The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims (4)

1. A primer for detecting bovine viral diarrhea virus is characterized in that: the primer is as follows:

5’-GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCCAUCCAACGAACUCACCACUGUUGCU-3’。

2. a kit for detecting bovine viral diarrhea virus, comprising:

comprises 45nM of purified LwCas13a protein, 22.5nM of specific crRNA, 125nM of quenched fluorescent reporter RNA, 2uL of RNase inhibitor and 40uL of total volume of detection buffer;

the specific crRNA is the primer of claim 1;

the quenching fluorescence reporter molecule RNA is as follows: 5'-GAAGAAGAGUUUAUUCAGAUAGAUUUGU-3' the flow of the air in the air conditioner,

the 5 'end is modified into FAM, and the 3' end is modified into BHQ-1.

3. The kit for detecting bovine viral diarrhea virus of claim 2, wherein:

the purification steps of the purified LwCas13a protein are as follows: constructing a LwCas13a prokaryotic expression vector, cloning the LwCas13a encoding gene to a prokaryotic expression vector pET-28a⁺Construction of pET-28a⁺-LwCas13a recombinant expression vector, after which pET-28a⁺-LwCas13a recombinant expression vector transferred into Rosetta^TM(DE3) expanding culture, inducing the expression of LwCas13a protein at low temperature, and obtaining purified LwCas13a protein by nickel column affinity chromatography.

4. A method for detecting bovine viral diarrhea virus based on CRISPR-Cas13a comprises the following steps:

constructing an LwCas13a prokaryotic expression vector;

step two, the LwCas13a is induced, expressed and purified;

designing specific crRNA and quenching fluorescent reporter RNA;

step four, establishing a CRISPR-Cas13a detection system, namely mixing the purified LwCas13a protein obtained in the step two, a designed crRNA sequence, a quenched fluorescent reporter RNA sequence, an RNase inhibitor and a detection buffer solution to obtain a mixture;

adding the sample to be detected into the mixture obtained in the step (iv), wherein detection of fluorescence indicates that the sample contains BVDV virus, and non-fluorescence indicates that the sample does not contain BVDV virus;

the steps of (I) and (II): the construction steps of the LwCas13a prokaryotic expression vector are as follows: cloning LwCas13a encoding gene to prokaryotic expression vector pET-28a⁺Construction of pET-28a⁺-LwCas13a recombinant expression vector, said pET-28a⁺-LwCas13a recombinant expression vector transferred into Rosetta^TM(DE3) carrying out amplification culture, inducing the LwCas13a protein to be expressed at low temperature, and obtaining purified LwCas13a protein by nickel column affinity chromatography;

step three: the sequence of the specific crRNA is as follows:

5’-GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCCAUCCAACGAACUCACCACUGUUGCU-3’；

the quenching fluorescence reporter molecule RNA sequence is:

5'-GAAGAAGAGUUUAUUCAGAUAGAUUUGU-3', modifying the 5 'end into FAM and the 3' end into BHQ-1;

adding a sample to be detected into the kit.

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